The invention relates to nucleotide sequences encoding bradykinin B1 receptors, recombinant expression vectors containing the genetic information, recombinant host cells capable of expressing the receptors and methods for identifying modulators of bradykinin B1 receptor activity.
The bradykinin (BK) B1 receptor, first identified in 1977, has been postulated to play a major role in the pathophysiology of chronic pain and inflammation. With some exceptions, most notably in several tissues in dog, the B1 receptor is not generally present unless its expression is induced by trauma or inflammatory stimuli. Following its induction, evidence suggests that the B1 receptor upregulates and maintains its own expression via an autocrine feedback loop upon occupation by its native ligands, des-Arg10-kallidin (dAKd) or, in the case of rodents, des-Arg9-BK (dABk). The autocrine nature of B1 receptor expression, coupled with the fact that the receptor is not generally expressed in normal, non-traumatized tissues, makes this an attractive target for anti-inflammatory and antinociceptive therapy.
In classical approaches to drug discovery, the activity of compounds are typically first analyzed by direct in vivo administration into test animals, or alternatively, in vitro using animal tissues. These avenues of discovery often lead to the identification of compounds potent in the test animal but of unknown efficacy in humans. In more recent paradigms of drug discovery, screening efforts are typically conducted on cloned human targets but resulting properties of lead candidates are sometimes complicated by lack of efficacy in animal models of choice.
The problem of potent but highly species-specific compounds is being encountered with greater frequency as the use of cloned human receptors in high-throughput drug screening becomes standard procedure. What is needed is an approach to address this problem by (1) looking at in vitro predictors of in vivo efficacy in animals and (2) screening molecules potent at the human receptor for activity at various animal orthologues in order to identify dually active compounds and an animal model useful in early efficacy studies of potential drug candidates.
In one aspect, the invention relates to a purified DNA molecule encoding a bradykinin B1 receptor from one of five mammalian species. The invention includes DNA molecules encoding an amino acid sequence chosen from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5 and functional derivatives thereof.
In another aspect, the invention relates to a purified DNA molecule having a nucleotide sequence chosen from SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10 and derivatives thereof.
In a related aspect, the invention relates to an expression vector which comprises a nucleotide encoding a bradykinin B1 receptor having a sequence chosen from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5 and functional derivatives thereof or a nucleotide having a sequence chosen from SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10 and derivatives thereof.
In yet another aspect, the invention relates to a recombinant cell comprising the expression vector described above and capable of expressing a bradykinin B1 receptor from the expression vector, as well as a method for producing such a cell. The method comprises transfecting a suitable host cell with the expression vectors described above and maintaining the host cells under conditions in which the bradykinin B1 gene is expressed.
In a related aspect, the present invention relates to a method of identifying a compound that modulates bradykinin B1 receptor activity. The method comprises contacting the test compound with a bradykinin B1 receptor comprising an amino acid sequence chosen from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5 and functional derivatives thereof; and measuring an effect of the compound on the receptor. The bradykinin B1 receptor may be expressed by a recombinant host cell. The effect to be measured may be a binding effect, for example, the displacement of a peptidic or non-peptidic ligand or a native ligand, such as, des-Arg10-kallidin, from the receptor.
In yet another aspect, the invention relates to a method of identifying an animal model for testing compounds with potential efficacy as bradykinin B1 receptor modulators. The method comprises contacting a test compound with a panel of bradykinin B1 receptors from several species; measuring an effect of the compound on the receptors; and then selecting an animal model for further study of the compound""s efficacy wherein the animal selected represents a species having a bradykinin B1 receptor that exhibits the desired effect when contacted with the test compound. In this method, the bradykinin B1 receptors comprise proteins having amino acid sequences chosen from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5 and functional derivatives thereof. Alternatively, the bradykinin B1 receptors are encoded by nucleotides having sequences chosen from SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10 and derivatives thereof.
In yet another aspect, the invention relates to a method of identifying a compound having dual specificity to modulate bradykinin B1 receptor activity in two different species comprising contacting the compound with a bradykinin B1 receptor from a first species; measuring an effect of the compound on said first receptor; contacting the compound with a bradykinin B1 receptor from a second species; measuring an effect of said compound on said second receptor; and, based on these measurements, determining whether the compound is equipotent for both receptors. In one embodiment, the method enables one to identify a compound having dual specificity to modulate bradykinin B1 receptor activity in a primate and non-primate.